Although the molecular target for drug therapy of sickle cell disease has been known for over 50 years, the only drug approved by the United States Food and Drug Administration is hydroxyurea. Hydroxyurea reduces the probability of vaso-occlusion by increasing the synthesis of fetal hemoglobin, which dilutes the abnormal hemoglobin S, markedly slowing its polymerization to form the fibers that distort (sickle) and make the red cells inflexible. This drug is, however, only partially successful in reducing the frequency of pain crises and the chronic organ damage characteristic of the disease. The search for additional and more effective therapeutic agents has been severely hampered by the lack of a sensitive assay for inhibition of sickling. We have developed a method to rapidly, accurately and sensitively test for anti-sickling activity in large populations of human red blood cells by measuring the distribution of sickling times. The first drug screen was carried out on the 2,000 compound library of Microsource Discovery, which contains 800 of the approximately 1,500 of unique compounds contained in the approximatelyy 3,500 FDA-approved drugs. The initial screen was carried out under hyperosmolar conditions. A second screen under iso-osmolar (i.e. physiological) conditions showed time-dependent effects for the most promising compounds. With technical improvements we now can make measurements at physiological osmolarity as a function of both time and concentration, and are re-screening a library of approximately 1,000 FDA approved compounds. The most promising compounds so far are those that reduce intracellular hemoglobin concentration by swelling red cells, taking advantage of the enormous concentration dependence of the delay time. We are also developing (1) assays to assess the effect of drug binding to plasma proteins, (ii) to test drugs on fractionated SS cells, and (iii) and very high thru-put assay for cell swelling. If the delay time is increased significantly at serum concentrations found for an FDA-approved compound, then, in principle, only two simple additional tests are required before clinical trials could begin, namely the hemolysis test and an oxygen binding curve to insure that the oxygen delivery function of the treated red cells is intact. Both of the assays are now fully developed. With this method we have shown that small increases in cell volume can result in therapeutic levels of fiber inhibition. We further show that one of the two anti-polymerization drugs currently in clinical trials that act by increasing oxygen affinity, also inhibits sickling in the absence of oxygen by increasing cell volume, potentially adding to its therapeutic benefit. We are now developing a truly high throughput screen based on nitrogen deoxygenation of sickle trait cells and a robust machine learning algorithm to determine the time at which an individual cell sickles.